LOCUS       sequence-491712-        6314 bp    DNA     circular SYN 04-NOV-2025
DEFINITION  CRISPR/Cas9 donor plasmid to tag human UBF with Halo tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6314)
  AUTHORS   Ide S, Imai R, Ochi H, Maeshima K
  TITLE     Transcriptional suppression of ribosomal DNA with phase separation.
  JOURNAL   Sci Adv. 2020 Oct 14;6(42). pii: 6/42/eabb5953. doi: 
            10.1126/sciadv.abb5953. Print 2020 Oct.
  PUBMED    33055158
REFERENCE   2  (bases 1 to 6314)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 4, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Sci Adv."; 
            date: "2020-10-14"; volume: "6(42). pii: 6/42/eabb5953. doi"; pages:
            " 10.1126/sciadv.abb5953. Print 2020 Oct"
COMMENT     Sequence Label: pGEM-HaloTag-UBF
FEATURES             Location/Qualifiers
     source          1..6314
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     111..130
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     regulatory      1045..1054
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      1049..1058
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(1088..1107)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1089..1107)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1114..1131)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1114..1130)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1123..1145)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1271..1726
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1358..1377)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1568..1589
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1804..1908
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1909..1977,1978..2769)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2127..2146)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2940..3528
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3429..3448
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3682..3699
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3816..3837
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3852..3857,3858..3875,3876..3882)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3890..3906
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     3895..3917
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3914..3930
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3914..3930
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        3948..3966
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     3948..3965
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             complement(4232..4243)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             complement(4888..5772)
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="EIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYV
                     WRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHD
                     WGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQ
                     NVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE
                     YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIG
                     SEIARWLSTLEISG"
     CDS             complement(5782..5838)
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             complement(join(5848..6314,1..130))
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(5928..5948)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
ORIGIN
        1 cgatgtggcg gtccgggtcg acggtgtggc gcgtggcggg gtagtcggcg aacgcggcgg
       61 cgagggtgcg tacggcccgg gggacgtcgt cgcgggtggc gaggcgcacc gtgggcttgt
      121 actcggtcat cctccagctg tccagccacc tcctcggtcg tgctggccgg gcaacccggg
      181 gtcaaagcca cctcaccctt tggaagacat accagttccc actcaggaag gctgagaggt
      241 gaacgactaa cgactttcta accgcccaga gtagaagggg gcaggtccac actctgagag
      301 actcagccat gaccttatta gacttaaggg gtctatggga atgggagtgc gcccctcctc
      361 ttcctcgtga ccccctcccc cagcccctgc agcccggctc cgcggcgcgc gccctcccct
      421 ggccacgcga gggcctgctc ctagctcccg cgtgcagcga gcgcccgccc tccgccccgc
      481 ttcccaaccc cagccaaaaa aaaaaaaaag aaaaaatctc ctcctcctcg gagcgcccgc
      541 cccggggagg ccccaggctg gagggcgggg gaagaggaag ggcgggtaaa gggcgcgcgc
      601 ggccacggaa gcgggcacgc gccccagtct cctcctcccg cctccccccg gccggttccc
      661 cccgcctccg caacccggcg gggacccgga cgcagcgcag ccgccgccag ccccggagcg
      721 gagcggccgc accgcccccg gggaccggat ccggattccc gccgctcccc cctcccgggc
      781 tcccttcgcc gtccccgccc gcacaggcgg gcaagcagcc ggagagaagc ggcctagcct
      841 gccccccgcc cccgagccac ccaccccggc cagaggcgcc gccaccgccc agccaccccg
      901 acgcgcacgg aggagcccgg cacagaggcg cgaccgcggc gggaaccgcc gcctcctccc
      961 ccgagcctgc ggcccagccc cgccggcgcc cccgccccgg cctccgggga gccgagccaa
     1021 ggagagggcg ggagaattcc cgcggccgcc atggcggccg ggagcatgcg acgtcgggcc
     1081 caattcgccc tatagtgagt cgtattacaa ttcactggcc gtcgttttac aacgtcgtga
     1141 ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag
     1201 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa
     1261 tggcgaatgg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     1321 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     1381 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     1441 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     1501 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     1561 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
     1621 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     1681 caaaaattta acgcgaattt taacaaaata ttaacgctta caatttcctg atgcggtatt
     1741 ttctccttac gcatctgtgc ggtatttcac accgcatcag gtggcacttt tcggggaaat
     1801 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     1861 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     1921 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     1981 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     2041 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     2101 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
     2161 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     2221 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     2281 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     2341 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     2401 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
     2461 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     2521 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     2581 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
     2641 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     2701 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     2761 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     2821 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     2881 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     2941 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     3001 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     3061 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     3121 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     3181 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     3241 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     3301 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     3361 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     3421 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     3481 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     3541 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     3601 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     3661 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata
     3721 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt
     3781 cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag
     3841 gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga
     3901 taacaatttc acacaggaaa cagctatgac catgattacg ccaagctatt taggtgacac
     3961 tatagaatac tcaagctatg catccaacgc gttgggagct ctcccatatg gtcgaccttg
     4021 tcagcccaac tacccaggga agcaagaaca ggtctggaac agttattttt cagccactga
     4081 aatgcagccc cagagagctc taggccactg tggatgggct gggagaatag ccacccaagg
     4141 ccagaagtca aaagggctct ccattttcat ttgcatctgg taacctgggc cagagattct
     4201 gcagaatggg ccacgtggac ccataatccc ctctcccctc aatgtcccac aagcatggcc
     4261 agctgtccag cttcatccta ctctctatcc ccatctagac tggatcactg ctgaatgagg
     4321 cagagatcta gttgagtaca tatatcccat ggcttggggc ctaaaaccac caaaacagcc
     4381 cttttctcca cctgcctcta caaaagcctc atggtcttcc taaaaagcct gagaagcctt
     4441 ccccaacccc ttcctgaccc ttcctctaca ctcatccttc ctcccattcc aggacactgg
     4501 gctgggacac acttcaggct gaatgggaac cccagaaggc aggcctcctc taacacagag
     4561 ggtgagggac agggagagag gggaggtgag ccactccagg ccaaataatt tggattcagc
     4621 caaaccccaa gtgtgggaga tgcttgattc ataaatactc aaggcacttt ctcccccagt
     4681 tcagtgtatg cagactgaaa aagcccttga agaagggagt gagccccgca gccacgaggg
     4741 aaagagggtt taagtttcag agggccgggg gtggatgccc ctaccttggc ctttgggggc
     4801 ggccatttcc aggtctgtgg ggcagtcggc ttctccgttc attgaaccgc cgccaccaga
     4861 tccacctcca ccagatccac ctccaccacc ggaaatctcc agagtagaca gccagcgcgc
     4921 gatctcgctg ccgatcaggt ccgggttgtc ttcttgcagc agattcagac ccgggccgat
     4981 gtccacagcc ttgcagttag gcaggctttt ggccaggcga gcggcttcgg ccggtgggat
     5041 cagaacgcct ggggtgcccc agaacagcag cttcgggaca ggggactggt gcagccagtc
     5101 catgtattct tcgaccagcg cgacgatgtt cgctggctca ccggcgattg gcagctcgtt
     5161 tgggaagcgc cacagtggct cgcggtcaac aggattcagg aacggctcgc ggtaatggtc
     5221 catctcgact tcagtcagcg ggcggacgac acccatcggc agcgtaccct cgataaaaac
     5281 gttctgatcg atgatcagct tgcggccgac gtcggtggtg cggaaggcct ggaaggtctc
     5341 gcgggcaaat tctggccatt cgtcccaggt cgggataggg cggatgaact ccataaatgc
     5401 aatacctttg acgcgctctg gattgcgctt ggcccagtgg aaacccagag cggagcccca
     5461 gtcgtgaatg accaggacga cctcttccag acccagggct tcgatgaagg catccatgaa
     5521 gcggacgtgg tcgtcgaaga aataacccag gtctggtttg tcggatttgc ccataccgat
     5581 caggtctgga gcaatgcagc gatgggtcgg tgcaacatgc gggatgatgt tgcgccacac
     5641 gtaggaggag gtcgggttac cgtgcaggaa cagcacaggg gtgccatcgc gcggaccaac
     5701 atcgacgtag tgcatgcgct cgcccaggac ttccacataa tgggggtcga atggaaagcc
     5761 agtaccgatt tcggatccca tgggaccggg gttttcttcc acgtctcctg cttgctttaa
     5821 cagagagaag ttcgtggctc cggatccggc accgggcttg cgggtcatgc accaggtgcg
     5881 cggtccttcg ggcacctcga cgtcggcggt gacggtgaag ccgagccgct cgtagaaggg
     5941 gaggttgcgg ggcgcggagg tctccaggaa ggcgggcacc ccggcgcgct cggccgcctc
     6001 cactccgggg agcacgacgg cgctgcccag acccttgccc tggtggtcgg gcgagacgcc
     6061 gacggtggcc aggaaccacg cgggctcctt gggccggtgc ggcgccagga ggccttccat
     6121 ctgttgctgc gcggccagcc gggaaccgct caactcggcc atgcgcgggc cgatctcggc
     6181 gaacaccgcc cccgcttcga cgctctccgg cgtggtccag accgccaccg cggcgccgtc
     6241 gtccgcgacc cacaccttgc cgatgtcgag cccgacgcgc gtgaggaaga gttcttgcag
     6301 ctcggtgacc cgct
//
